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BACKGROUND: Secondhand smoke (SHS) contains more than 7000 chemicals, 70of which are carcinogens and it is estimated that every one in four Americans areinvoluntarily exposed to SHS. Carcinogenesis induced by SHS exposure isunclear, though SHS exposure is associated with various cancers including headand neck squamous cell carcinoma (HNSCC). Recently, it has been shown thatSHS exposure is a significant independent predictor of HNSCC recurrence. Wehave shown that in vitro exposure to SHS increases HNSCC cisplatin resistance. AIMS: Our study examines the effect of SHS smoke exposure on cisplatin-induced HNSCC cancer cell death and the possible mechanisms behind thisobservation. METHODS: Sidestream smoke (SS), the main component of SHS,was extracted as previously described. Authenticated HNSCC cell lines (UM-SCC1 & WSU-HN6) were exposed to SS extract for 48 hours at doses mimickingthe nicotine levels observed in the saliva of passive smokers. Then, cancer cellswere treated with cisplatin in the presence or absence of SS extract. Cisplatin-induced cancer cell death was measured using Incucyte® S3 Live-Cell imaginganalysis. Transcriptomic analysis following RNA-sequencing was performedusing Ingenuity Pathway Analysis. Data were subjected to ANOVA. RESULTS: Our live-cell imaging system revealed that HNSCC cells exposed to SS extractsignificantly decreased cisplatin-induced cancer cell death at various time points compared with cells treated with cisplatin alone in both cell lines. The HNSCCcells treated only with SS extract did not show any change in cell death at 0, 12,24, 48, 72, and 96 hours. RNA-sequencing data revealed that cell death andsurvival pathways are the most changed pathways in the SS extract-treatedgroup compared with unexposed HNSCC cells. CONCLUSIONS: Our studydocuments for the first time that even short-term exposure to SHS allows cells toevade cisplatin-induced cell death and can lead to head and neck cancercisplatin resistance. At the molecular level, SHS exposure alters genes that areassociated with cell death and survival. Further, HNSCC patients-basedobservations and mechanistic studies are warranted. FUNDING: This work was partially supported by the National Cancer Institute of the NationalInstitutes of Health (R33CA202898, R01CA242168, and P30CA225520), theTSET Health Promotion Cancer Center, and the Oklahoma Center forAdvancement of Science and Technology (HR16-007). Dr. Queimado holds aPresbyterian Health Foundation Endowed Chair in Otorhinolaryngology Position. Citation Format: Balaji Sadhasivam, Jimmy Manyanga, Vengatesh Ganapathy, Célia Bouharati, David Rubenstein, Jonathan Wren, Pawan Acharya, Daniel Zhao, Lurdes Queimado. Secondhand smoke exposure alters cisplatin-induced cell death and alters cell death and survival pathway in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 384.

Background: E-cigarette use has skyrocketed among youth, in part because e-cigarettes are perceived as a safer substitute for traditional cigarettes. However, the resulting health effects of e-cigarette use are still unclear. E-cigarette aerosols contain harmful and potentially harmful substances such as flavorings, carbonyl compounds, heavy metals, carcinogens and reactive oxygen species (ROS). A recent in vitro study from our laboratory showed that e-cigarette aerosols can increase cellular ROS and suppress cellular antioxidant capacity. An imbalance between the ROS production and the availability of antioxidants or free radical scavengers, can lead to chronic inflammation. Here we examine the effect of exposure to e-cigarette aerosols on the expression of the inflammatory regulator NF-kB and its downstream inflammatory targets in oral epithelial cells. Methods: Human oral epithelial cancer (UM-SCC-1) cells were exposed, every other day for 2 weeks, to e-cigarette aerosol extracts (18 mg/ml of nicotine; tobacco flavor) prepared from two distinct e-cigarette brands. Standard tobacco extracts were used as a positive control. Whole-cell RNA was isolated and processed for RNA-sequencing. The altered gene expression was further validated by western blotting and ELISA using a human cytokine array. Data were analyzed by Student's t-test. Results: RNA sequencing data showed that a 2-week exposure of oral epithelial cells to e-cigarette aerosol extracts resulted in significant changes in several key cellular signaling pathways, including inflammatory and immune response signals. Exposure to e-cigarette aerosol extracts resulted in a significant increase in the expression of proteins involved in inflammatory pathways, such as TLR3, TGF beta, ERK1/2 and NF-kB. We also observed a significant increase in pro-inflammatory and anti-inflammatory cytokines after exposure to e-cigarette aerosol extracts, including CD54, IL-1a and IL-10. Conclusion: Our data suggest that chronic exposure to e-cigarette aerosol increases the expression of TLR3, TGF beta, and ERK1/2, which possibly contributed to the observed increase in NF-kB and its down-stream targets (e.g., CD54, IL-1a and IL-10). NF-kB is a pleiotropic transcription factor with key roles in multiple biological processes, such as immune homeostasis and development. Chronic NF-kB activation by e-cigarette aerosol exposure could contribute to chronic inflammation and tumorigenesis. Citation Format: Vengatesh Ganapathy, Jimmy Manyanga, Dehra McGuire, Daniel Brobst, Balaji Sadhasivam, Mayilvanan Chinnaiyan, Constantin Georgescu, Jonathan Wren, Lurdes Queimado. E-cigarette aerosol exposure increases NF-kB and modulates inflammatory markers in oral epithelial cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4470.

Results that are statistically significant are more likely to be reported by authors and more likely to be accepted by journals. These common biases warp the published evidence and undermine the ability of research to improve health by giving an incomplete body of evidence. We aimed to show the effect of the bias towards statistical significance on the evidence-base using published confidence intervals. We examined over 968,000 ratios and their confidence intervals in the abstracts of health and medical journals from Medline between 1976 and January 2019. We plotted the distributions of lower and upper confidence interval limits to visually show the strong bias for statistically significant results. There was a striking change in the distributions around 1, which is the statistically significant threshold for ratios. There was an excess of lower intervals just above 1, corresponding to a statistically significant increase in risk. There was a similar excess of upper intervals just below 1, corresponding to a statistically significant decrease in risk. These biases have not improved in recent years. The huge excesses of confidence intervals that are just above and below the statistically significant threshold are not statistically plausible. Large changes in research practice are needed to provide more results that better reflect the truth.

INTRODUCTION Sjögren’s syndrome (SS) is a rheumatic autoimmune disease characterized by focal lymphocytic infiltrates in the lacrimal and salivary glands, severe dry mouth and eyes, pain and debilitation. Diagnosis requires autoantibodies to ubiquitous Ro antigens or a lip biopsy positive for focal lymphocytic infiltrates. Here we used human proteome arrays to identify novel antibodies in plasma from Ro positive and Ro/La antibody negative SS patients compared with healthy controls. METHODS Anti-Ro positive (n=15) and anti-Ro negative (n=15) cases meeting 2016 ACR/EULAR classification criteria for SS were age, race, and sex matched with each other and healthy controls (n=15). Plasma IgG binding to human proteome arrays containing >19,500 recombinant human proteins representing >80% of the human proteome (HuProt v3.2 arrays, CDI Laboratories) was assessed. Data were normalized by the Robust Linear Model using the PAA Bioconductor Package in R and log intensity values for each protein generated. Thresholds of mean + 4SD were established using the controls. Antigens bound by IgG more frequently in cases compared to controls (p RESULTS IgG from Ro positive SS cases significantly bound 18 proteins, including the canonical SS antigens Ro60 and Ro52. IgG from Ro negative SS cases significantly bound 4 proteins compared to controls, 3 of which were shared with the Ro positive group. Binding to any one of 4 novel proteins identified 73% of the Ro negative SS cases. CONCLUSION A total of 17 novel antigen specificities were identified in SS, with 4 antigens being bound by plasma IgG from Ro/La negative SS cases. These antigens may be useful for diagnosing SS without a lip biopsy.

Prostate cancer (PCa) is a complex heterogeneous disease, with the majority of cases remaining indolent and 10% of cases progressing to lethality. While some of this clinical heterogeneity can be explained by traditional clinicopathological factors, molecular profiling studies signal an extraordinary genomic variability, which may lead to diverse clinical outcomes. The identification of molecular subclasses of PCa has the potential to guide the prediction of clinical outcomes, the discovery and design of innovative prognostic biomarkers, and the development of novel therapeutics. To this end, we investigated genes associated with TMEFF2, an androgen-regulated tumor suppressor gene with exceptionally heterogeneous expression in PCa. Low levels of TMEFF2 mRNA significantly (p Citation Format: Joshua M. Corbin, Constantin Georgescu, Sandra Thibivilliers, Zachary Webb, Yan Zhao, Jan Koster, Kar-Ming Fung, Adam Asch, Jonathan Wren, Maria Ruiz-Echevarria. A tumor suppressor-regulated cell cycle derived gene signature is prognostic of recurrence risk in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3152.

Introduction Cancer associated thrombosis is the second most common cause of mortality among patients with malignancy. While preventive low molecular weight heparin is effective in preventing venous thromboembolism (VTE), lack of agreement on risk stratification methods have precluded its widespread use. The most studied VTE prediction tool is the Khorana score, but it does not capitalize on the use of novel biomarkers. We aimed to evaluate the value of biomarkers in prediction of VTE among patients with active cancer receiving outpatient chemotherapy. Methods We prospectively collected pre-chemotherapy blood samples on adult patients with solid organ tumors. We excluded patients on anticoagulation and those unable or unwilling to consent. Along with demographics and cancer specific variables, potential novel predictors of VTE were selected a priori and included: coated platelets (CP), D dimer (DD), protein C (PC), fibrin monomers (FM), mean platelet volume (MPV), platelet microparticles (PMP). Type of cancer was grouped according to the Khorana score (Very high risk: Stomach, Pancreas; High risk: Lung, Gynecologic, Bladder, Testicular; Low/Moderate risk: all other sites). VTE events were adjudicated at 6 months of follow up. For all statistical analyses we used SAS 9.2 (SAS Institute Inc., Cary, NC, USA). A p value of Results We have recruited a total of 101 patients (mean + standard deviation age 59.7+11 years, 68% female), 20% of the 75 patients with complete follow up have developed a VTE. We found no significant association between the incidence of VTE and age, gender, or baseline Khorana score. The variables associated with incident VTE were: BMI (odds ratio [OR] 8.5 for BMI Conclusion Pre Chemotherapy level of Protein C is a potential biomarker for the incidence of VTE. We confirm that cancer type and BMI are predictors of cancer associated VTE. In addition, lower BMI is not usually recognized as a potential predictor of VTE in other prediction scores. We were underpowered to detect differences in the VTE incidence according to Khorana score. Finally, aspirin may have a role in outpatient cancer associated VTE prevention and deserves further investigation. Disclosures: Tafur: Stago Diagnostica: Research Funding. Rathbun:Stago Diagnostica: Research Funding.

Genetic polymorphism in MECP2/IRAK1 is a confirmed susceptibility locus for lupus. DNA methylation changes in T cells play a central role in the pathogenesis of lupus, and MeCp-2 is a master regulator of gene expression and is known to recruit DNA methyltransferase 1 (DNMT1) during DNA synthesis. Using human T cells from normal individuals, we demonstrate that the lupus risk variant in MECP2/IRAK1 increases MECP2 mRNA expression in stimulated T cells. By assessing DNA methylation levels at ~ 485,000 methylation sites across the genome, we further show that the lupus risk variant in this locus is associated with significant DNA methylation changes, including in the HLA-DR and HLA-DQ loci, as well as interferon-related genes such as IFI6, IRF6, and BST2. Further, using a human MECP2 transgenic mouse, we show that overexpression of MECP2 alters gene expression in stimulated T cells. This includes overexpression of Eif2c2 that regulates the expression of multiple microRNAs (such as miR-21), and the histone demethylase Jhdm1d. We also show that MECP2 transgenic mice develop antinuclear antibodies. Our data suggest that the lupus associated variant in the MECP2/IRAK1 locus has the potential to affect all 3 epigenetic mechanisms. Importantly, we demonstrate that variants within the MECP2 gene can alter DNA methylation in other genetic loci including the HLA and interferon-regulated genes, thereby providing evidence for genetic-epigenetic interaction in lupus.

BANK1, an adaptor protein expressed in B-cells, is genetically associated with human SLE and plays a little understood role in B-cell signaling. Because BANK1 contains an N-terminal putative Toll-interleukin 1 receptor (TIR) domain, we used murine Bank1-/- splenic B cells to test if BANK1 affects signaling induced by the TLR9 agonist CpG. Following CpG stimulation BANK1 deficiency reduced p38 phosphorylation without affecting that of ERK or JNK and to reduced IL-6 secretion. Bank1-/- cells showed reduced phosphorylation of MNK1/2, confirming that BANK1 affects p38 signaling, and expression microarray analysis showed a disregulated type I interferon response. Microarray analysis and reduced phosphorylation of eIF4E indicated general repression of the eIF4F translation initiation complex in Bank1-/- cells. Together these data establish previously unrecognized roles for BANK1 in CpG-induced responses by splenic B cells. BANK1 may regulate type I IFN responses to TLR9 stimulation by influencing p38 signaling and the eIF4F complex, and thereby influence the course of viral infections or autoimmunity.

In inflammation, elevated GM-CSF directs the development of dendritic cells (DCs) from myeloid precursors that infiltrate tissues. These inflammatory DCs play a central role in infection and autoimmunity. We previously showed that estradiol and estrogen receptor alpha (ERα) act in murine Flt3+ myeloid progenitors to promote GM-CSF-mediated DC differentiation. ERs are ligand-dependent transcription factors that form complexes with chromatin-modifying coregulators near gene promoters. Thus, we hypothesized that ERα regulates chromatin structure at loci important for DC differentiation. We used anti-ERα chromatin immunoprecipitation and next generation sequencing to identify genome-wide sites of ERα binding in myeloid progenitors stimulated with GM-CSF -/+ estradiol. Bioinformatics analyses identified unique ERα binding sites in the presence of estradiol near the transcription start site of >900 genes. Binding of ERα was enriched at CpG islands and consensus binding sites for SP1 and GABPA transcription factors. ERα bound to genes in pathways related to ER, GM-CSF and ERK/MAPK signaling, protein ubiquitination and cell cycle progression. ERα also bound to multiple genes in the NF-κB and Toll-like receptor pathways. These data suggest that ERα regulates genes involved in the NF-κB and GM-CSF signaling pathways during GM-CSF-mediated differentiation, and primes myeloid progenitors or mature DCs for responses to inflammatory stimuli.

Abnormal T cell DNA methylation and type-1 interferon play an important role in the pathogenesis of systemic lupus erythematosus. We performed a genome-wide DNA methylation study in two independent sets of lupus patients and matched healthy controls to characterize the DNA methylome in naïve CD4+ T cells in lupus. DNA methylation was quantified for over 485,000 methylation sites across the genome, and differentially methylated sites between lupus patients and controls were identified and then independently replicated. Gene expression analysis was also performed from the same cells to investigate the relationship between the DNA methylation changes observed and mRNA expression levels. We identified and replicated 86 differentially methylated CG sites between patients and controls in 47 genes. Importantly, we observed significant hypomethylation in interferon-regulated genes in naïve T cells from lupus patients, suggesting epigenetic transcriptional accessibility of these genetic loci. The hypomethylation in interferon-regulated genes was not related to lupus disease activity. Gene expression analysis showed overexpression of these genes in total but not naïve CD4+ T cells from lupus patients. Our data suggest epigenetic “poising” of interferon-regulated genes in lupus naïve CD4+ T cells, argue for a novel pathogenic implication for abnormal T cell DNA methylation in lupus, and suggest a mechanism for type-1 interferon hyper-responsiveness in lupus T cells.

Determining the most effective parameters for in vitro propagation of T cell precursors is critical for realizing the promise of many envisioned T cell-based immunotherapies. To this end, the consequences of culturing ETP and DN2 thymocytes with IL-18 and IL-1β in the context of the OP9-DL4 co-culture model were evaluated after inference of a hypothesized role for these molecules in thymopoiesis based on results obtained via a novel microarray/literature mining algorithm. Sort-purified mouse ETPs and DN2 thymocytes were cultured on OP9-DL4 stromal layers ±IL-18, IL-1β, IL-12 and/or IL-7 for one week. IL-18 and IL-12 alone promoted proliferation and differentiation of ETPs to the DN3a stage and these cytokines could compensate for absence of IL-7. When IL-18 was used in conjunction with IL-7, synergistic proliferation was observed. IL-1β proved to be deleterious to development and was not overcome by inclusion of IL-7. To evaluate the developmental potential of the derived pre-T cells, culture-generated DN3a-like cells were sort-purified then cultured for an additional 8 days. In these assays, the pre-T cells demonstrated differentiation to the DP stage. Overall, these results indicate that IL-18 and IL-12 can positively impact T cell development and should prove useful in strategies to augment in vitro T cell development in the OP9-DL4 system.